Hypoxia-inducible factor-1 (HIF-1) is the primary regulator of the transcriptional response of mammalian cells to low oxygen and other stresses that lower ATP levels. Our investigations on the mechanism of action of HIF-1 have led us to the fundamental observation that adenosine 5'-monophosphate-activated protein kinase (AMPK) activity is important for tumor biology. AMPK is strongly activated in both wild type (wt) and genetically matched cells lacking HIF-1 activity (HIF-1 null cells) under conditions of hypoxia and glucose deprivation, indicating that activation of AMPK is a response to hypoxia that is distinguishable from HIF-1 action. This raises the possibility that AMPK activity may differ in solid tumor cells compared to normal cells due to differing metabolic states. To investigate this possibility, we examined the growth of tumor xenografts from genetically manipulated mouse embryo fibroblasts (MEFs) lacking expression of AMPK catalytic 1 subunits (AMPK null cells) and found that growth of the AMPK null tumors was strikingly suppressed compared with that of the wt tumors. Based on this and additional in vitro and in vivo observations, we hypothesize that AMPK activity is a therapeutic target in solid tumors. Currently no compounds are publicly available to validate the suitability of AMPK as a target for chemotherapy, however. The ligand used most often in studies of AMPK inhibition is Compound C, for example, which is more potent as an inhibitor of the VEGF receptor (KDR) than of AMPK. This selectivity profile is clearly inadequate to discriminate in vivo mechanisms of action in cancer models since KDR is itself a target for chemotherapy. We propose to use structure-based drug design to identify small molecule inhibitors that bind selectively with high affinity to AMPK. The approach will include the design and synthesis of lead allosteric inhibitors of AMPK (allosteric inhibitors are chosen to maximize selectivity) and co-crystallization of these leads with AMPK to generate protein structures that will enable further optimization of the leads. These compounds will be used to establish proof of concept for AMPK as a therapeutic target in solid tumors, first in animal models and finally in the clinic - the ultimate goal and commercial application. [unreadable] [unreadable] [unreadable]